Molecular Phenotyping of Patients with Sepsis and Kidney Injury and Differential Response to Fluid Resuscitation

Purpose Previous work has identified two AKI sub-phenotypes (SP1 and SP2) characterized by differences in inflammation and endothelial dysfunction. Here we identify these sub-phenotypes using biospecimens collected in the emergency department and test for differential response to restrictive versus liberal fluid strategy in sepsis-induced hypotension in the CLOVERS trial. Methods We applied a previously validated 3-biomarker model using plasma angiopietin-1 and 2, and soluble tumor necrosis factor receptor-1 to classify sub-phenotypes in patients with kidney dysfunction (AKI or end-stage kidney disease [ESKD]). We also compared a de novo latent class analysis (LCA) to the 3-biomarker based sub-phenotypes. Kaplan-Meier estimates were used to test for differences in outcomes and sub-phenotype by treatment interaction. Results Among 1289 patients, 846 had kidney dysfunction on enrollment and the 3-variable prediction model identified 605 as SP1 and 241 as SP2. The optimal LCA model identified two sub-phenotypes with high correlation with the 3-biomarker model (Cohen’s Kappa 0.8). The risk of 28 and 90-day mortality was greater in SP2 relative to SP1 independent of AKI stage and SOFA scores. Patients with SP2, characterized by more severe endothelial injury and inflammation, had a reduction in 28-day mortality with a restrictive fluid strategy versus a liberal fluid strategy (26% vs 41%), while patients with SP1 had no difference in 28-day mortality (10% vs 11%) (p-value-for-interaction = 0.03). Conclusion Sub-phenotypes can be identified in the emergency department that respond differently to fluid strategy in sepsis. Identification of these sub-phenotypes could inform a precision-guided therapeutic approach for patients with sepsis-induced hypotension and kidney injury.


INTRODUCTION
Acute kidney injury (AKI) is the most common form of organ failure in sepsis developing in as many as 60% in septic patients with hypotension 1,2 .Sepsis-associated AKI is associated with prolonged hospitalization, need for kidney replacement therapy, future chronic kidney disease (CKD) and death 3- 8 .At present, providers base treatment decisions in AKI on parameters such as urine output and serum creatinine, yet these biomarkers fail to differentiate the highly heterogenous clinical syndrome of AKI and cannot reliably predict response to common interventions such as volume resuscitation and vasopressors in sepsis 9,10 .
We previously applied latent class analysis (LCA) to two cohorts to identify two distinct sub-phenotypes in patients with sepsis-associated AKI, and subsequently developed a parsimonious 3-variable model for clinical application 11 .This 3-variable model included plasma biomarkers of endothelial function (angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2)) and in ammation (soluble tumor necrosis factor receptor-1 [sTNFR-1]) 11 .Ang-1 and -2 are vascular endothelial growth factors that have opposing activities.Ang-1 is an agonist for the Tie-2 receptor and is protective by stabilizing the endothelium, promoting vessel maturation, and preventing microcirculatory capillary leakage.In contrast, Ang-2 typically acts as an antagonist for binding to the Tie-2 receptor and promotes endothelial dysfunction in the form of vessel destabilization, endothelial cell apoptosis, capillary leak, and deregulation of in ammation [12][13][14] .Independently, sTNFR-1 functions in the early innate in ammatory response in sepsis and has been associated with AKI and mortality 15 .Our model established two distinct AKI subphenotypes: those with a lower ratio of Ang-2/Ang-1 and low sTNFR-1, designated SP1 and characterized by endothelial stability and improved short and long-term outcomes.In contrast, those with higher ratio of Ang-2/Ang-1 and high sTNFR-1, or SP2 phenotype, were characterized by high in ammatory state and leaky endothelium and worse clinical outcomes 11,[16][17][18] .
The Crystalloid Liberal or Vasopressors Early Resuscitation in Sepsis (CLOVERS) clinical trial randomized patients with sepsis to an initial restrictive or liberal uid resuscitation strategy and demonstrated no differences in clinical outcomes between treatment arms or in the sub-groups with AKI or end-stage kidney disease (ESKD) 19 .Early and aggressive uid resuscitation is a cornerstone of sepsis management which aims to improve hypoperfusion caused by increased capillary leak and endothelial permeability.In the setting of kidney injury or disease, however, the bene t of uid resuscitation is counterbalanced by impaired volume management.We hypothesized that liberal uid resuscitation would be preferentially harmful to septic patients with evidence of both endothelial injury and kidney disease, and that application of the SP1 or SP2 sub phenotypes may enable identi cation of patients most likely to bene t from early vasoconstrictive therapy.To address these questions, we rst identi ed sub-phenotypes with known kidney injury (AKI or ESKD) using plasma samples collected in the emergency department, representing an early and critical time for treatment implementation in sepsis.Second, in a sensitivity analysis, we veri ed sub-phenotypes by applying LCA to 23 variables collected at study enrollment and compared sub-phenotype identi cation to the 3-variable model.Third, since serum creatinine can lag up to 48 hours and is insensitive for kidney injury 20 , we conducted a sensitivity analysis including patients without AKI on study enrollment to test whether the treatment effect by sub-phenotype was preserved.Fourth, to understand the overlap and differences between different sub-phenotypes in critical illness, we cross-tabulated our sub-phenotypes with a previously established hypo-and hyperin ammatory acute respiratory distress syndrome (ARDS) sub-phenotypes.

Study Design and Oversight
We conducted a secondary analysis of data from CLOVERS, a multicenter, prospective, phase 3, randomized, non-blinded trial.Patients enrolled in CLOVERS were allocated to either a restrictive or liberal uid resuscitation strategy in the setting of sepsis.All patients were randomized within 4 hours of meeting inclusion criteria, and duration of protocol for restrictive or liberal uid resuscitation was 24 hours.Patients randomized to a restrictive uid protocol had vasopressors prioritized as the primary treatment for sepsis-induced hypotension, with "rescue uids" being permitted for prespeci ed indications that suggested severe intravascular volume depletion.The liberal uid protocol consisted of a recommended initial 2 L IV infusion of isotonic crystalloid, followed by uid boluses administered based on clinical triggers with "rescue vasopressors" permitted for prespeci ed indications.A more detailed description of methods can be found in the Supplement of the primary manuscript 19 .

AKI De nition
AKI was de ned using the KDIGO criteria as an increase in serum creatinine at randomization of ≥50% or ≥0.3 mg/dL above a baseline serum creatinine.The lowest outpatient or inpatient serum creatinine value within the last year prior to the index hospitalization was used for the baseline when available (N=909).If no pre-hospitalization serum creatinine was available and there was no known history of CKD, then we imputed a baseline serum creatinine based on an eGFR of 90 ml/min/1.73m2 (N=517) using the 2021 creatinine-based CKD-EPI equation 11 .If no pre-hospitalization serum creatinine was available and there was a known history of CKD, then we used the lowest serum creatinine value during the index hospitalization as the baseline (N=29).Patients were subsequently staged according to the serum creatinine at randomization using the KDIGO guidelines.Seventy-ve patients were on maintenance hemodialysis prior to study enrollment, (ESKD).Since ESKD patients represent severe impairment in kidney-mediated volume management and guidelines do not offer guidance on distinct therapeutic approaches across the spectrum of ESKD, we included them in the analysis.

AKI Sub-phenotype Identi cation
Methods for measurement of Ang-1, Ang-2 and sTNFR-1 can be found in the online supplement.We used plasma biomarker concentrations of Ang-1, Ang-2 and sTNFR-1 to identify two sub-phenotypes (SP1 and SP2).Since the publication of our prior works, Meso Scale Discovery changed the sTNFR-1 assay from a R-Plex to a U-Plex platform.In analyses provided in the supplement we found the correlation of these two assays to be high (Pearson's correlation, r = 0.94), and we created a calibration equation (Figure S1).We updated the 3-variable prediction model that initially used R-Plex measured sTNFR-1 to include U-Plex measured values to account for the updated sTNFR-1 biomarker platform.The model to identify AKI sub-phenotypes is TNFR_R-Plex = -101.3387+0.3617*TNFR_U-Plex followed by the previously published prediction equation Logit(P(SP2)) = -35.44+1.99*log(Ang-2/Ang-1)+ 3.41*log(TNFR_R-Plex).Participants with a probability of SP2 > 0.5 were then classi ed as having the SP2 sub-phenotype.
Methods of LCA applied to the baseline variables can be found in the Methods in the supplement.Previously described ARDS sub-phenotypes were identi ed using a published four variable model that included plasma concentrations of interleukin-6, sTNFR-1, serum bicarbonate and clinical documentation of receipt of vasopressors prior to randomization 11,21 .

Outcome Measures
The primary outcome for this analysis was 28-day and 90-day mortality.Additional outcomes included need for invasive mechanical ventilation (IMV), need for new KRT, and ICU-free days through 28 days of follow-up.

Statistical Analysis
We compared the risk of death by sub-phenotype over 28 and 90 days of follow-up using Cox regression, adjusting in nested models for potential confounders.To test for heterogeneity of treatment effect, we completed an intention-to-treat comparison of the treatment effect of restrictive or liberal uid resuscitation strategy on 28-day and 90-day mortality by sub-phenotype.For this analysis, we used Kaplan-Meier 28-day and 90-day mortality point estimates involving all patients who were discharged home or were still alive at day 90, with data censored at day 91.Patients who were lost to follow-up prior to day 90 were censored at the time they were last con rmed to be alive.We compared 28-day and 90day mortality point estimates and evaluated the evidence for a treatment by sub-phenotype interaction using a non-parametric bootstrap approach with 10,000 replicates.
Secondary outcomes were the time to IMV among patients who were not receiving IMV at randomization and the time to new KRT among patients not on dialysis at the time of randomization.For these outcomes, we used a Fine-Gray sub-distribution hazard model to estimate the sub-distribution hazard ratio of each outcome accounting for the competing risk of death.Finally, we used linear regression to estimate the difference in ICU-free days through day 28 between the two treatment groups.All analyses were conducted using the R 4.2.3 software environment (R Foundation for Statistical Computing, Vienna, Austria).Two-sided p-values < 0.05 were taken as evidence of statistical signi cance.

RESULTS
Baseline Characteristics of Participants by Sepsis-associated AKI Sub-phenotype Among 1563 patients enrolled in CLOVERS, 1289 had biospecimens collected prior to randomization and available for analysis (Figure 1).Among the 1289 patients, 846 (66%) had kidney dysfunction (771 had AKI and 75 had ESKD) at study enrollment and 443 (34%) did not have AKI or ESKD.Among the 846 patients with kidney dysfunction the previously published sub-phenotype classi cation model, incorporating plasma Ang-1, Ang-2, and sTNFR-1 measurements at randomization, classi ed 605 (72%) patients as SP1 and 241 (28%) as SP2 (Table 1).As expected by randomization, the distribution of SP1 and SP2 were similar between treatment arms.Patients with SP2 had more severe KDIGO stage of AKI on study enrollment compared to SP1 (Figure S2).
To determine whether SP2 is enriched in the AKI population, we applied the 3-biomarker prediction model to patients without AKI or ESKD on study enrollment.Among 443 patients, we found that 411 (93%) were classi ed as SP1 and 32 (7%) as SP2.The 32 patients with SP2 were characterized by high rates of CKD and ten patients subsequently developed AKI within 48 hours of study enrollment based on changes in serum creatinine (Table S1).In a sensitivity analysis, we applied LCA to 23-variables collected at study enrollment and found that a 2-class model best separated the data (Table S3).We also found high correlation between the LCA derived sub-phenotypes and the 3-variable biomarker sub-phenotypes, Cohen's Kappa of 0.80.With the high correlation and to support prospective identi cation of these subphenotypes, we used the 3-variable derived sub-phenotypes for the subsequent analyses.

Clinical Outcomes by Sub-Phenotype
To evaluate the association of sub-phenotypes with clinical outcomes, patients without AKI were compared against those with SP1 and SP2 and then patients with SP1 and SP2 were directly compared (Table S4).In models adjusting for demographics, comorbidities, KDIGO stage of AKI or ESKD and baseline sequential organ failure assessment (SOFA) scores, patients with SP1 had a similar risk of new KRT, ICU length of stay, receipt of IMV, 28-day and 90-day mortality as patients with no AKI.Patients with SP2 had greater risk of 28-day (HR = 2.33 (95% CI: 1.60, 3.38) and 90-day mortality (HR = 1.98 (95% CI: 1.45, 2.71) than SP1.Patients with SP2 were more likely than those with SP1 to require new KRT (sHR=3.03(95% CI: 1.42, 6.47) or IMV after study enrollment (sHR=1.54(95% CI: 1.00, 2.37) (Table S5).Those with SP2 had ICU stays that were 3 days longer than those with SP1 (adjusted difference 3.1 days (95% CI: 1.8 to 4.5 days) (Table S7).

Difference in Volume of Fluid and Timing of Vasopressors by CLOVERS Randomization
In the 24-hour protocol following randomization, patients with SP1 randomized to the liberal resuscitation strategy received on average 2 L more of IV uids than patients with SP1 randomized to the restrictive resuscitation strategy (3,684 ± 1,644 mL vs 1,684 ± 1657 mL) (Table S6).Similarly, patients with SP2 randomized to the liberal resuscitation strategy received on average 4,091 (± 1793) mL compared to 2,244 (± 2,305) mL in the restrictive strategy.Consistent with the trial protocol, patients randomized to the restrictive strategy received vasopressors more often than patients in the liberal strategy across both sub phenotypes.

Interaction Between Sub-phenotype and Resuscitation Strategy
Next, we determined whether sub-phenotypes identi ed using biospecimens collected prior to randomization respond differently to the uid resuscitation strategy.In SP1, a similar proportion of patients died at 28 days with liberal and restrictive uid strategies (11% vs 10%, respectively) (Figure 2).In SP2 a liberal strategy compared with a restrictive strategy led to greater 28-day mortality (41% vs 26% respectively) and the difference in treatment effects between SP1 and SP2 was 14% (95% CI: 2%, 27%); p-value for interaction = 0.03.This effect was not observed in the patients without AKI (Table 2).Among patients with SP1, 90-day mortality with a liberal and restrictive uid strategies was 18% versus 17%, while SP2 was 48% versus 26% (p-value for interaction = 0.06).We did not observe a difference for alternative outcomes among sub-phenotypes by treatment (Table S8).Next, we evaluated the continuous probability of belonging to SP2 and the interaction of a liberal versus a restrictive uid resuscitation.We observed a signi cant interaction between the probability of sub-phenotype and uid resuscitation strategy and 28-day mortality (p=0.008)(Figure 3).As patients had a greater certainty of the SP2 phenotype (i.e. higher probability of SP2 based on the 3-biomarker model), we observed a larger difference in 28-day mortality between a liberal versus restrictive uid resuscitation strategy.In the primary CLOVERS publication, there was no observed heterogeneity of treatment effect based on SOFA scores or ESKD status 19 .
In sensitivity analyses, we evaluated sub-phenotypes restricted to the AKI population and excluded ESKD.
Among patients with SP1, 28-day mortality with liberal and restrictive uid strategies was 10% versus 10% and in patients with SP2 was 39% versus 29% (p-value for interaction = 0.16) (Table S9).In another sensitivity analysis, we used the 3-biomarker model to classify all patients with plasma available in CLOVERS as SP1 and SP2.Among patients with SP1, 28-day mortality with a liberal and restrictive uid strategies was 9% versus 9% and in patients with SP2 was 41% versus 27% (p-value for interaction = 0.02).We also observed a similar decrease in 90-day mortality in SP2 (Table 2).

Cross-Tabulation of AKI and ARDS Sub-phenotypes
We directly compared the cross-tabulation of SP1 and SP2 and ARDS sub-phenotypes (hypoin ammatory and hyperin ammatory).Cross-tabulation demonstrated mild to moderate overlap between hypoin ammatory and SP1 and hyperin ammatory and SP2 but also demonstrated distinct differences (Cohen's Kappa for Agreement of 0.42) (Table S10).Out of 846 patients, 77.0% were concordant (i.e.hypoin ammatory and SP1 or hyperin ammatory and SP2), and 23.0% were classi ed discordantly.We observed no differential response to a restrictive or liberal uid resuscitation strategy between ARDS sub-phenotypes for 28-day (p-value for interaction = 0.25) or 90-day mortality (p-value for interaction = 0.60) (Table S11).

DISCUSSION
The heterogeneity of AKI has limited insight into clinical trajectory and hindered development of interventions that target an individual patient's pathophysiology [22][23][24][25] .We completed a secondary analysis of the CLOVERS trial and demonstrated that sub-phenotypes previously de ned in ICU patients were also present in the emergency department, an earlier and critical window for therapeutic decisions in sepsis treatment.These sub-phenotypes predicted differential treatment response to liberal versus restrictive resuscitation strategies among patients with kidney dysfunction; revealing a treatment effect which was not seen in the original CLOVERS study.Taken together, these data offer a precision medicine treatment strategy in sepsis-induced hypotension and kidney dysfunction based on a 3-biomarker prediction model which may identify populations with differential response to uid resuscitation strategy.
Despite a long-held belief by clinicians that a rise in serum creatinine early during sepsis should result in additional uid therapy 9 , patients with SP2 and kidney dysfunction had a greater mortality with a liberal uid strategy compared to a restrictive uid strategy.A difference in clinical outcomes between uid strategy was not observed in patients without AKI/ESKD or patients with SP1.While these ndings are hypothesis-generating, it suggests that early in sepsis the combination of kidney dysfunction and endothelial injury may identify a sub-phenotype that is harmed by a liberal uid therapy.In addition, the observed heterogeneity of treatment effect was not present when identifying sub-groups based on SOFA scores, ESKD or AKI status 26,27 , suggesting that the sub-phenotypes capture information that is not readily available by clinical de nitions.In this manner, application of the 3-biomarker prediction model to all patients with sepsis-induced hypotension may improve generalizability and time to sub-phenotype identi cation to facilitate future clinical trials without misclassi cation of patients with SP2.
Our prior work leveraged these AKI sub-phenotypes in the Vasopressin and Septic Shock Trial (VASST), a randomized control trial investigating early addition of vasopressin to norepinephrine in sepsis.In that analysis, we showed that in SP1, early addition of vasopressin compared to norepinephrine alone was associated with improved 90-day mortality, but in SP2, vasopressin showed no signi cant treatment difference.Extrapolating these ndings with the current CLOVERS analysis, we hypothesize that patients with the SP1 would derive bene t from either a restrictive or liberal uid resuscitation and early addition of vasopressin with norepinephrine for persistent hypotension, while those with the SP2 may bene t from an initial restrictive uid strategy and monotherapy with norepinephrine.
Our study has several strengths.First, a majority of patients had a pre-hospitalization serum creatinine value available to approximate baseline kidney function and to accurately determine AKI status early after hospital presentation.Leveraging this data highlighted that approximately two-thirds of patients in the trial had AKI on hospital presentation for sepsis-induced hypotension.These ndings are consistent with prior studies of AKI in septic shock and highlight that prevention of AKI in sepsis may be infeasible and therapies that target recovery should be prioritized 28,29 .Second, we were able to leverage the randomization in the CLOVERS trial to address limitations of indication bias of uid administration in observational studies to demonstrate that patients with SP2, characterized by greater endothelial dysfunction and in ammation, have an improved mortality with a restrictive resuscitation strategy compared to a liberal strategy.Third, the CLOVERS trial included patients with ESKD, a group that is often excluded from large randomized clinical trials 30 .While limited by small group size, our data suggest that most patients with ESKD have a sub-phenotype consistent with SP2 (79%) and may bene t from a restrictive uid resuscitation strategy.This analysis should be interpreted in the context of its limitations.First, there was variation in the amount of uid received in the CLOVERS trial in the restrictive and liberal uid groups and overlap of total volumes between groups may dampen a potential therapeutic signal.Second, this was a retrospective study of a completed clinical trial.However, the study design and prediction model for sub-phenotype identi cation were de ned a priori.Third, we underscore that our work does not preclude that alternative AKI sub-phenotypes are present.Fourth, elevations of angiopoietins and sTNFR-1 are not speci c to AKI.However, a large body of preclinical and clinical studies have demonstrated the importance of these pathways and biomarkers in kidney diseases, such as AKI and CKD 13,31,32 .Fifth, serum creatinine concentrations were not collected after day 3 of participation in CLOVERS and so we are unable to draw conclusions about differential rate of kidney recovery between sub-phenotypes.
In summary, this work posits that early identi cation of AKI sub-phenotypes can inform clinical decision making in patients with sepsis-induced hypotension.We observed that SP2 had improved 28-day mortality with a restrictive uid resuscitation strategy.